ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY OF LICHENS ...

Malaysian Journal of Analytical Sciences, Vol 20 No 1 (2016): 1 - 13

1

MALAYSIAN JOURNAL OF ANALYTICAL SCIENCES

Published by The Malaysian Analytical Sciences Society

ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY OF LICHENS Usnea

rubrotincta, Ramalina dumeticola, Cladonia verticillata AND THEIR

CHEMICAL CONSTITUENTS

(Kajian Aktiviti Antibakteria dan Antioksidan Liken Usnea rubrotincta, Ramalina dumeticola,

Cladonia verticillata dan Sebatian Kimianya)

Saranyapiriya Gunasekaran1*, Vinoshene Pillai Rajan

1, Surash Ramanathan

2, Vikneswaran Murugaiyah

3,

Mohd. Wahid Samsudin1, Laily B Din1

1School of Chemical Science and Food Technology, Faculty of Science and Technology

Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia 2Centre for Drug Research

3School of Pharmaceutical Sciences

Universiti Sains Malaysia, 11800 Gelugur, Penang, Malaysia

*Corresponding author: [email protected]

Received: 24 August 2015; Accepted: 4 November 2015

Abstract

The present study was carried out to evaluate the antibacterial and antioxidant activity of extract and chemical constituents of

Usnea rubrotincta, Ramalina dumeticola and Cladonia verticillata. Acetone extract of U. rubrotincta showed promising

antibacterial activity against Gram positive bacteria Bacillus subtilis with the lowest Minimal Inhibitory Concentration (MIC)

value of (15.63 µg/mL). Six secondary metabolites were isolated using Preparative High Performance Liquid Chromatography

(PHPLC) method from the two bioactive lichens U. rubrotincta and R. dumeticola (compound 1 – 6). Among all six compounds,

compound (1) exhibited strongest activity against both the tested Gram positive bacteria at 7.81 µg/mL. Compound (6) had

69.57% scavenging activity against DPPH free radical while the rest only showed below 50% scavenging activity. This is the

first evaluation of antibacterial activity of lichens found in Malaysia and to our knowledge, this is the first report of antibacterial

and antioxidant activity of compound (3) and (5).

Keywords: antibacterial, antioxidant, Usnea rubrotincta, Ramalina dumeticola, Cladonia verticillata, usnic acid

Abstrak

Kajian ini dijalankan bagi menilai aktiviti antibakteria dan antioksidan sebatian kimia liken yang diekstrak iaitu Usnea

rubrotincta, Ramalina dumeticola dan Cladonia verticillata. Ekstrak aseton terhadap U. rubrotincta telah menunjukkan aktiviti

antibakteria yang memberangsangkan terhadap bakteria Gram positif Bacillus subtilis dengan nilai Kepekatan Perencatan

Minimum (KPM) sebanyak (15.63 µg/mL). Enam metabolit sekunder telah dipencilkan daripada 2 liken bioaktif iaitu U.

rubrotincta dan R. dumeticola menggunakan kaedah Kromatografi Cecair Prestasi Tinggi Preparatif (KCPTP) (sebatian 1 – 6).

Daripada 6 sebatian, sebatian (1) telah memaparkan aktiviti yang paling kuat terhadap kedua-dua bakteria Gram positif yang

dikaji pada kepekatan 7.81 µg/mL. Sebatian (6) mempunyai peratus pemerangkapan radikal bebas DPPH pada 69.57% manakala

sebatian lain hanya menunjukkan aktiviti pemerangkapan kurang daripada 50%. Ini adalah penilaian pertama terhadap aktiviti

antibakteria bagi liken yang dijumpai di Malaysia dan kepada pengetahuan kita, ini adalah laporan pertama bagi aktiviti

antibakteria dan antioksidan sebatian (3) dan (5).

Kata kunci: antibakteria, antioksidan, Usnea rubrotincta, Ramalina dumeticola, Cladonia verticillata, asid usnik

ISSN

1394 - 2506

Saranyapiriya et al: ANTIBACTERIAL AND ANTIOXIDANT ACTIVITY OF LICHENS Usnea rubrotincta,

Ramalina dumeticola, Cladonia verticillata AND THEIR CHEMICAL CONSTITUENTS

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Introduction

Lichens are self-supporting symbiotic association between a mycobiont and a photobiont. The mycobiont which is

the fungus is unique and dominates the association while the photobiont is the algae or cyanobacteria. Many lichens

are able to endure extreme environmental conditions hostile to the survival of the individual partners [1]. They are

able to grow as scattered patches on stones, outcrops, on tree trunks or shrubs. Lichens mostly expose their

vegetative parts to sunlight to enable the photobiont which harvest energy from solar radiation. These vegetative

bodies are called thalli or lichenized stroma which contains characteristic secondary compounds [2].

To date approximately 1050 secondary metabolites have been identified [3] and most of them are considered to be

very unique. The secondary metabolites found in lichens were reported to have multiple functions. They are light

filters that protect the photobionts from excessive radiation [4], prevent damage from grazing by herbivores and

have antibiotic properties to protect against microbial degradation [5]. Compounds like atranorin,

fumarprotocetraric acid, gyrophoric acid, lecanoric acid, physodic acid, protocetraric acid, stictic acid and usnic acid

have showed comparatively strong antimicrobial properties against bacteria and fungi, amongst which were human,

animal and plant pathogens, mycotoxin-producers and food-spoilage organisms [6]. Several depsides (sekikaic acid,

lecanoric acid and lobaric acid) are known exhibited radical scavenging activity comparable to rutin [7].

There are compelling reasons for the search of new antimicrobial agents, including from natural product drugs

mainly because of the emergence of multi drug-resistant pathogens and the reduced effectiveness of currently used

drugs. Besides, the rise of various diseases related to oxidative stress implicates the dire need of discovery of new

antioxidant agents. Lichens can be a good source for the search due to the ability to survive in extreme conditions.

Malaysia is abundant of highland and lowland lichens. Din et al. [8] profiled several types of chemical compounds

derived from lichens collected from Bukit Larut, Peninsular Malaysia some of which have been reported previously

to possess strong antibacterial and antioxidant activity. Four lichen species namely Ramalina peruviana,

Parmotrema tinctorum, Bulbothrix isidiza and Cladia aggregata from Penang Hill, Malaysia were found to have

significant antioxidant activity [9]. Hitherto there is no report on the antimicrobial activity of the lichens found in

Malaysia. This study investigated antibacterial and antioxidant potential of various extracts of three different lichens

from the species, Usnea rubrotincta, Ramalina dumeticola and Cladonia vercillata collected from the Malaysian

highland area. Further to this, work was undertaken to isolate the chemical constituents from the active lichen

extracts.

Materials and Methods

Lichen samples

Lichen Ramalina dumeticola was collected from Fraser Hill, Pahang, 3° 46 N’ 111° 43 E’, 1300m above sea level,

voucher specimen number (F.H 1). Lichen Usnea rubrotincta and Cladonia vercilata were collected from Bukit

Larut, Perak, 4° 50N’, 100° 48 E’, 1035m above sea level, voucher specimen number (B.L 1, B.L 2). Voucher

specimens of these lichens have been deposited at the School of Chemical Sciences and Food Technology,

Universiti Kebangsaan Malaysia.

Microorganisms and media

The bacteria used as test organisms are as follows: Staphylococcus aureus (ATCC 29213), Bacillus subtilis (ATCC

19659) and Escherichia coli (ATCC 25922) obtained from American Type Culture Collection/USA). Bacterial

cultures were maintained on Muller Hinton agar substrates (Oxoid Ltd., Hampshire, England) and were stored at 4 oC.

Preparation of lichen extracts

The lichen samples were air dried at room temperature. Powdered lichen samples of 250 g were subjected to

sequential soxhlet extraction with two types of solvent, acetone and methanol. All the extractions were repeated

twice. The extracts were then filtered, concentrated in vacuo and air-dried under fume hood to obtain dry powdered

extracts which were stored at 4°C.


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Antibacterial studies

The minimal inhibitory concentrations (MIC) of methanol and acetone extract of the lichens were determined using

the 96 well plate methods as described by Eloff [10] with some modification. Bacterial inoculums were obtained

from bacterial cultures incubated for 24 hours at 37 °C on Müller-Hinton agar and diluted to 0.5 McFarland

standard of approximately 108 CFU/mL. The extracts were screened for antibacterial potentials at 500 µg/mL.

Extracts showing bacterial growth inhibition were further tested to evaluate its MIC. A two fold serial dilution of

acetone extracts (500 – 7.81 µg/mL) in DMSO and methanol extracts in methanol were prepared in a sterile 96-well

plate respectively. The percentage of solvent was kept below 1% throughout the experiment.

Bacterial suspensions (100 mL) were added to each well and the plates were incubated for 24 hours at 37 °C. After

the incubation time, 50 mL of p-iodonitrotetrazolium (p-INT) (0.2 mg/mL) was added into all the wells, including

the blank wells (containing methanol or DMSO), and further incubated for 1 hour. These plates were then observed

for color changes. Wells with yellow color were interpreted as no growth, whilst purple color was interpreted as

presence growth. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of

samples which inhibit any visible growth of bacteria. Chloramphenicol and vancomycin were used as positive

controls (500 – 7.81 µg/mL). All experiments were performed in triplicate.

Antioxidant assay: Total phenolic content

Total phenolic content (TPC) were estimated by using the Folin – Ciocalteu method [11] with some modification.

Stock solutions of 1 mg/mL extracts and gallic acid were prepared in DMSO and distilled water respectively. A

series of gallic acid concentrations were prepared by diluting the stock solution with distilled water. Gallic acid of

250 µL was added into individual test tubes followed by addition of 1 mL of distilled water and 250 µL of Folin –

Ciocalteu reagent. After thoroughly shaken, the test tubes are allowed to sit in room temperature for 6 minutes.

Later, an amount of 2.5 mL of Na2CO3 was added and allowed to sit for another 2 hours in a dark room at room

temperature. A 200 µL of solution from each test tube is transferred into 96-well plate and the absorbance were

measured at 760 nm using a microplate reader. The experiments were performed in triplicates. Gallic acid (0–

500 µg/mL) was used for calibration of a standard curve. The result was expressed as gallic acid equivalents

(GAE)/g dry weight of lichen material.

Total flavonoids

The total flavonoid content (TFC) was determined using colorimetric method as described by Sakanaka et al. [12].

An amount 125 µL of 1 mg/mL extract or standard solution (quercetin) were mixed with 625 µL of distilled water

in a test tube. Sodium nitrite (5%) solution of 37.5 µL is then added and incubated for 6 minutes. Aluminium

chloride (10%) solution of 75 µL was later added and the mixture was allowed to stand for another 5 minutes.

Thereafter, 250 µL of 1M sodium hydroxide was added. The mixture was brought up to 1.25 mL with distilled

water and mixed well. The absorbance at 510 nm was measured immediately. Quercetin (0.5–0.02 mg/mL) was

used to construct a standard curve. The results were expressed as quercetin equivalent (QE) per gram dry weight of

lichen extract. The experiments were performed in triplicates.

1, 1-diphenyl-2-picryl-hydrazyl (DPPH) free radical analysis

A quantitative determination of free radical scavenging activity was measured by using DPPH assay as described by

Shimada et al. [13]. The antioxidant activity was expressed as percentage scavenging of DPPH by extract compared

to standard, Trolox. An amount of 20 µL extract (1 mg/mL) was added into 180 µL of 0.2 mM of DPPH solution in

96-well plate. The plate was incubated for 20 minutes and kept in the dark. The same steps were repeated for

standard Trolox. The percentage of scavenging was calculated from the absorbance of decolorization which

obtained spectrophotometrically at 550 nm. Scavenging of the DPPH radical by the sample was calculated

according to the following Equation (1).

DPPH scavenging activity (%) = [A0 – A1 / A1] × 100 (1)

where A0 is the absorbance of the control and A1 is the absorbance of the sample. Free radical scavenging activity is

expressed as the percentage of DPPH scavenging activity. The experiments were performed in triplicates.



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Isolation of chemical constituents from the bioactive extracts: Liquid-liquid extraction

2 g of bioactive extracts were subjected to liquid-liquid extraction using hexane and methanol at 1:1 ratio. The

process was performed in a triplicate. The resulting fractions were pooled and dried. It was noticed that there were

crystals formed, which was filtered and purified further by recrystallization.

Thin layer chromatography (TLC)

The fractions were dissolved in appropriate solvent before being spotted onto the TLC plate. Solvent systems used

in this thin-layer chromatography were according to Culberson [14] and Culberson et al. [15] namely (a) benzene-

dioxane-acetic acid (180:45:5, 230 mL), (b) hexane-diethyl ether-formic acid (130:80:20, 230 mL), (c) toluene-

acetic acid (200:30, 230 mL) and (d) toluene-ethyl acetate-formic acid (139:83:8, 230 mL). The spots were viewed

under long and short ultraviolet light before sprayed with 10% sulphuric acid and heated at 110 °C.

Column chromatography

Fractions that showed good separation on the TLC plate was subjected to column chromatography. The solvent

system used was similar to the one used for TLC. An approximately 0.5 g of the fraction was dissolved in a small

amount of solvent system to avoid solubility problems inside the column. Small amounts of solvent were filled into

the column every 15 minutes. The sub-fractions were collected according to the bands formed in the column and air-

dried before subjected to TLC.

Preparative high performance liquid chromatography

The column used for separation was a Phenomenex Hypersil 5μ C18 column (150 x 21.2 mm) with the gradient

mobile phases of methanol (A) and 1% orthophosphoric acid (B) with a flow rate of 4 mL/min. The run started with

100% B and was lowered to 50% B within 5 minutes then to 40% B within a further 10 minutes, followed by 20% B

within the next 30 minutes. Then 500 µg/ml of sample with the injection volume of 100 μl was injected. A

photodiode array detector was used to detect the compound peaks and recorded at three different wavelengths of

222 nm, 230 nm and 280 nm. The fractions were collected according to the retention time.

Identification of chemical constituents

An analytical HPLC method adapted from Din et al. [8] was used to evaluate the purity of the compounds obtained.

A Phenomenex Hypersil 3μ C18 column (250 by 4.6 mm) with a flow rate of 1mL/min was used. The mobile

phases were 1% aqueous orthophosphoric acid (A) and methanol (B). The gradient system started with 100% A and

was raised to 58% B within 15 minutes, then to 100% B within next 16 minutes, followed by isocratic elution in

100% B for a further 10 minutes. The compounds were further subjected for mass (LC-MS ToF), 1D NMR FT-

NMR 600MHz Cryo (Fourier Transform Nuclear Magnetic Resonance 600MHz Cryoprobe) and 1D NMR FT-

NMR 500MHz (Fourier Transform Nuclear Magnetic Resonance 500MHz) spectrometry.

Bioassay for isolated chemical constituents

The isolated chemical constituents were evaluated for antibacterial and antioxidant activity at 500 μg/mL using the

methods described in section Antibacterial studies and Antioxidant assay. The active chemical constituents were

further tested for their (MIC).

Results and Discussion

Chemical Constituents

Usnic acid, (1) was obtained as yellow needles from acetone extract of U. rubrotincta, (1190 mg, 6.62% yield);

HPLC retention time: 28.76 minutes; Mass spectrometry data showed 345 m/z; 1H NMR data and

13C NMR data,

see Table 1; Structure, see Figure 1.

Barbatic acid, (2) was obtained as colorless needles from methanol extract of U. rubrotincta, (0.13 mg, 0.31%

yield); HPLC retention time: 29.02 minutes; Mass spectrometry data showed 360m/z;

1H NMR data and

13C NMR

data, see Table 2; Structure, see Figure 1.


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Table 1. 1H and

13C NMR spectral data (CDCl3) for compound (1)

Analysis δH δC

NMR

C-1 198.06

C-2 179.39

C-3 - s, 1H, OH-3 155.22

C-4 5.986 s, 1H, H-4 191.73

C-5 101.53

C-6 98.35

C-7 109.33

C-8 13.320 s, 1H, OH-8 157.51

C-9 103.97

C-10 11.039 s, 1H, OH-10 163.88

C-11 105.24

C-12 59.08

C-13 1.766 s, 3H, Me-13 27.93

C-14 200.37

C-15 2.669 s, 3H, Me-15 32.14

C-16 2.111 s, 3H, Me-16 7.56

C-17 201.80

C-18 2.684 s, 3H, Me-18 31.31

Notes: Chemical shift values δ in ppm.

Table 2. 1H and


Analysis δH δC

C-1 110.17

C-2 152.23

C-3 104.25

C-4 3.930 s, 3H, MeO-4 162.32

C-5 6.559 s, 1H, H-5 106.19

C-6 140.16

C-7 170.01

C-8 2.069 s, 3H, Me-8 6.53

C-9 2.706 s, 3H, Me-9 23.59

C-4-OMe 54.78

C-1’ 162.27

C-2' 110.50

C-3’ 115.98

C-4’ 162.77

C-5’ 6.575 s, 1H, H-5’ 140.75

C-6' 115.70

C-7' 173.77

C-8’ 2.054 s, 3H, Me -8' 7.99

C-9' 2.595 s, 3H, Me-9' 24.30

- s, 1H, 2-OH




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(1) (2)

(3) (4)

(5) (6)

Figure 1. Chemical structure of (1) Usnic acid, (2) Barbatic acid, (3) 8-Hydroxybarbatic acid, (4)

Atranorin, (5) Hyperhomosekikaic acid and (6) Sekikaic acid


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8-Hydroxybarbatic acid, (3) was obtained as colorless crystals from methanol extract of U. rurbotincta, (5.18 mg,

0.01% yield); HPLC retention time: 28.94 minutes; Mass spectrometry data showed 391.29 m/z;

1H NMR data see

Table 3; Structure, see Figure 1.

Atranorin, (4) were obtained as colorless prisms from acetone extracts of R. dumeticola, (51.28 mg, 0.29% yield);

HPLC retention time: 29.05 minutes; Mass spectrometry data showed 373.11 m/z;

1H NMR data and

13C NMR data,

see Table 4; Structure, see Figure 1.

Table 3. 1H NMR spectral data (CDCl3) for compound (3)

Analysis δH

1.185 s, 3H, Me

2.023, 2.031 s, 3H, Me

2.104 s, 3H, Me

2.522

2.556

2.618

3.424 s, 3H, OMe-4

3.836 s, 2H, -CH2OH-8

6.312 s, 1H, H-5

6.478 s, 1H, H-5’

11.424 s, 1H, 1-OH

11.872 s, 1H, 1-OH


Table 4. 1H and


Analysis δH δC

C-1 102.901

C-2 12.503 s, 1H, OH-2 169.165

C-3 2.678 s, 3H, Me-9 108.606

C-4 12.551 s, 1H, OH-4 167.552

C-5 6.392 s, 3H, Me-5 112.926

C-6 152.527

C-7 169.776

C-8 10.349 s, 1H, CHO-8 193.932

C-9 24.130

C-1’ 116.849

C-2’ 11.962 s, 1H, OH-2’ 162.949

C-3’ 110.318

C-4’ 152.037

C-5’ 6.507 s, 1H, C-5’ 116.091

C-6’ 139.956

C-7’ 172.285

C-8’ 2.080 s, 3H, Me-8’ 25.681

C-9’ 2.536 s, 3H, Me-9’ 9.456

COOMe-7’ 3.969, 3.979 s, 3H, COOMe-7’ 52.444

- 29.787




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Hyperhomosekikaic acid, (5) were obtained as colorless plates from acetone extract of R. dumeticola, (4.92 mg,

0.20% yield); HPLC retention time: 27.87 minutes; Mass spectrometry data showed 480 m/z;

1H NMR data see

Table 5; Structure, see Figure 1.

Sekikaic acid, (6) were obtained as colorless prisms from acetone extracts of R. dumeticola, (4.73 mg, 0.20% yield);

HPLC retention time: 29.45 minutes; Mass spectrometry data showed 406 m/z; 1H NMR data see Table 6;

Structure, see Figure 1.


Analysis δH

NMR

0.913, 0.926, 0.938 t, 3H, Me-5’’

0.985, 0.997, 1.009 t, 3H, Me-5’’’

1.128, 1.138 m, 6H, 3x -CH2

1.290, 1.302, 1.314

2.158

2.93, 2.94, 2.96 t, 2H, -CH2- 1”

2.98, 2.99, 3.009 t, 2H, -CH2- 1”’

3.82 s, 3H, MeO- 4

3.876 s, 3H, MeO- 4’

6.365, 6.369 d, 1H, H-3’

6.393, 6.397 d, 1H, H-5’

6.548 s, 1H, H- 5



Analysis δH

NMR

0.823, 0.838, 0.852 t, 3H, Me

1.195, 1.212, 1.227 t, 3H, Me

1.288, 1.295, 1.302, 1.316 -

1.547, 1.561 q, 2H, -CH2

1.608, 1.623, 1.639, 1.654 q, 2H, -CH2

2.843, 2.859, 2.874 t, 2H, -CH2-1”

2.909, 2.925, 2.941 t, 2H, -CH2-1”’

3.343, 3.347, 3.350

3.730 s, 3H, -OMe

3.784 s, 3H, -OMe

6.274, 6.279 d, 2H, H-3

6.302, 6.308 d, 2H, H-5

6.448 s, 1H, H-5’



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Bioassay of extracts

The antibacterial activity and the (MIC) of extracts are summarized in Tables 7 and 8 respectively. Acetone extracts

of Usnea rubrotincta and Ramalina dumeticola and methanol extracts of Usnea rubrotincta exhibited inhibitory

activity against gram positive bacteria S. aureus and B. subtilis. However no activity against E. coli, gram negative

bacterium was observed. The remaining lichen extracts were not active against the three test bacteria. The acetone

extract of R. dumeticola exhibited highest activity against S. aureus followed by acetone and methanol extracts of

U. rubrotincta. For negative control, DMSO did not exhibit any inhibition against all test organisms.

Table 7. Antibacterial activity of acetone and methanol extracts of various lichens at 500 µg/mL concentration

Test

Organisms

Usnea

rubrotincta

Ramalina

dumeticola

Cladonia

verticillata

Chloramphenicol Vancomycin

A M A M A M

S. aureus + + + - - - + +

B. subtilis + + + - - - + +

E. coli - - - - - - + +

Notes: A, acetone extract; M, methanol extract; +, had inhibitory activity against tested bacteria; -, had no inhibitory activity of

extract against tested bacteria

Table 8. Minimal Inhibitory Concentration of bioactive extracts against selected gram positive bacteria

Test Organisms Usnea rubrotincta Ramalina dumeticola Chloramphenicol Vancomycin

A M A

Minimal Inhibitory Concentration (µg/mL)

S. aureus 125 500 31.25 31.25 7.81

B. subtilis 15.63 250 31.25 15.63 7.81

Notes: A, acetone extract; M, methanol extract

Both acetone and methanol extracts of U. rubrotincta showed activity against gram positive bacteria. The lowest

MIC (15.63 µg/mL) was observed for acetone extract of U. rubrotincta against B. subtilis comparable to

chloramphenicol. A similar activity against B. subtilis and S. aureus was reported for Usnea barbata acetone extract

with MIC value of 0.1 mg/mL [16]. R. dumeticola acetone extract showed good antimicrobial activity against both

gram positive bacteria with MIC values though it was approximately four times less potent than vancomycin (Table

8). Lichen from the same genus, Ramalina farinacea was also reported active against some gram positive bacteria

including B. subtilis and S. aureus at MIC value of 264 µg/mL and 132 µg/mL respectively [17]. All the lichen

extracts in this study showed no activity against gram negative bacteria. These findings are closely in agreement

with report by Burkholder et al. [18] whom reported no activity of 42 tested lichens against gram negative bacteria

E.coli.

The phenol and flavonoid contents of all three lichens were in the range of 43.12 – 101.62 mg of GAE/g extract and

24.92 – 165.03 mg of QE/g extract respectively. The DPPH assay showed scavenging activity lower than 50% of for

all the studied lichens at 500 µg/mL. Results are summarized in (Table 9).



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Table 9. Total phenolic content (TPC), Total flavonoid content (TPC) and DPPH radical scavenging activity

of the lichen extracts

Sample TPC

(mg) of (GAE)/g dry

weight of extract

TFC

mg of (QE)/g dry

weight of extract

% Scavenging of DPPH

at 500µg/mL

Acetone RD 101.62 ± 3.51 24.92 ±1.06 27.21 ± 1.61

U.R 63.05 ± 3.27 165.03 ±0.62 19.31 ± 0.25

C.V 101.37 ± 3.31 108.09 ±3.95 33.09 ± 0.84

Methanol

R.D 48.74 ± 0.84 42.77 ± 1.13 16.4 ± 2.99

U.R 43.12 ± 1.14 98.48 ± 2.13 11.42 ± 1.96

C.V 48.61 ± 0.87 79.20 ± 1.65 17.25 ± 2.98

Trolox 98.19 ± 0.66

Another study [19] recorded (TPC: 115mg of (GAE)/g dry weight of extract) and (TFC: 1.625mg of rutin

equivalent/g dry weight of extract) values of a different species of Usnea lichen which is Usnea longissima. The

same study also reported high antioxidant activity (IC50 = 0.10 mg dry material). Phenolic compounds are a type of

antioxidant agents which can adsorb and neutralize the free radicals [20], in which the phenol content in present

study is lower than reported study [19]. This explains the low antioxidant activity of U. rubrotincta compared to U.

longissima. Cladonia foliacea was investigated for its TPC and DPPH scavenging activity [21], which also reported

low phenolic content (TPC: 78.12mg of (GAE)/g dry weight of extract) and weak scavenging activity at (IC50: 1000

µg/mL) which is in accordance with the present study. Lichen from the genus Ramalina, Ramalina peruviana was

investigated for its phenolic content, as well as its free radical scavenging activity [9]. They also reported lowest

total phenolic content in R. peruviana (27.1 mg GAE/g extract) compared to a few other lichen species studied,

however the free radical scavenging activity was found to be relatively strong at (IC50 = 60.66 mg dry material). R.

dumeticola was found to contain higher phenolic content but the free radical scavenging activity was only 27.21%.

This can be due to different types and concentrations of phenolic compounds present in R. peruviana and R.

dumeticola.

Since the antioxidant activity of the extracts was moderate compared to the antibacterial activity, the extracts

selected for subsequent purification and isolation was based on the antibacterial assay results. A total of six

secondary metabolites were isolated from acetone and methanol extract of R. dumeticola and U. rubrotincta.

Compound (1) and (4) were found in both the lichen acetone extracts which were identified as usnic acid and

atranorin respectively. Compounds (5) and (6) were isolated from acetone extract of R. dumeticola which was

determined to be hyperhomosekikaic acid and sekikaic acid. With regards to the methanol extract of U. rubrotincta,

two more compounds were isolated which were (3), 8-hydroxybarbatic acid and (2), barbatic acid.

Among all isolated chemical constituents, (1) exhibited highest inhibitory activity against both gram positive

bacteria at (MIC: 7.81 µg/mL) which is comparable to standard vancomycin. Atranorin and (2) also showed active

inhibitory potential at (MIC: 31.25 µg/mL) and (MIC: 15.63 µg/mL) against B. subtilis. The high antimicrobial

activity of (1) has been reported previously by Tay et al. [17] in which the lowest inhibitory concentration against S.

aureus was at (MIC: 3.1 μg/62.5 μl) and B. subtilis at (MIC: 0.78 μg/62.5 μl). Compound (1) has been observed to

be present in high levels in both the active lichens which could partly explain the potency of the extracts comparable

to the standard.

Lauterwein et al. [22] reported a MIC value range from 2 to 16 μg/mL for (1) against gram positive bacteria S.

aureus. In this study, compound (4) has exhibited stronger activity than the report by Yilmaz et al. [23], where the

inhibition potential was only at (MIC: 500 µg) against S. aureus and (MIC: 15.63 µg) against B. subtilis. Thadani et

al. [24] reported similar activity of (4) and (6) against B. subtilis with inhibition at 100 µg/mL in a disk diffusion

assay.


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However, they also reported inhibition activity of (4) and (6) against gram negative bacteria E. coli and no inhibition

of (4) and (6) against S. aureus. Both reports by Yilmaz et al. [23] and Thadhani et al. [24] have significant

difference with this study due to method difference where the reported studies used disk diffusion assay compared to

this study which uses the MIC method. Besides that, the bacteria strains are also different between this investigation

and Yilmaz et al. [23]. Martins et al. [25] reported MIC values of 50-100 µg/mL for (2) against various strains of S.

aureus which is similar to this study where we obtained MIC value of 62.50 µg/mL concentration. Disk diffusion

method has its own disadvantages where zone sizes are affected by the media used and by the different growth rates

of organisms being tested therefore MIC tests are more accurate with anaerobic bacteria [26].

All compounds exhibited below 50% scavenging activity against DPPH free radicals except compound (6). Sisodia

et al. [27] reported close activity between the standard and compound (6) against DPPH free radical but in this study

only 69.57% scavenging activity was recorded compared to standard Trolox. They also reported minimum

scavenging activity of (4) in which is in agreement with this study. Compound (1) which is a phenolic compound,

poorly scavenged the DPPH free radical which is in accordance with the findings of Devehat et al. [28] and

Thadhani et al. [7]. The radical-scavenging effect of antioxidants on DPPH is a method to quantify the hydrogen

donating potency of chemicals and compound (1) does not seem to have the labile hydrogen atoms to bind with the

DPPH ions. Devehat et al. [28] studied the antioxidant capacity of compound (2) alongside other lichen compounds

but did not report any scavenging activity which is in agreement with our findings where the activity was also very

low in contrast to standard. Results are summarized in Table 10.

Table 10. Antibacterial and radical scavenging activity of the isolated compounds

Test Compounds MIC (µg/mL) % DPPH radical scavenging

at 100μg/ml

S. aureus B. subtilis

(1) 7.81 7.81 44.62 ± 0.22

(2) 62.50 31.25 29.29 ± 0.003

(3) - 125 36.60 ± 0.001

(4) 62.50 15.63 27.79 ± 0.11

(5) - 125 13.25 ± 0.002

(6) - 125 69.57 ± 0.52

Chloramphenicol 31.25 15.63

Vancomycin 7.81 7.81

Trolox 98.41 ± 0.01

Notes: - mean no activity detected against tested bacteria

Conclusion

This is the first report on biological study in of lichens Usnea rubrotincta, Ramalina dumeticola, and Cladonia

verticillata from Malaysia. Among the chemical constituents isolated in this study, 8-hydroxybarbatic acid from U.

rubrotincta and hyperhomosekikaic acid from R. dumeticola has been investigated for its antibacterial and

antioxidant capacity for the first time and were found to be considerably active against tested bacteria. Usnic acid

and atranorin have been identified as active antibacterial agents while sekikaic acid as a considerable antioxidant

agent. Further work should be done to enhance the knowledge on these chemical constituents.



12

Acknowledgement

This research was supported by Exploratory Research Grant Scheme (ERGS/1/2012/STG01/UKM/01/3), Ministry

of Higher Education, Malaysia, Universiti Kebangsaan Malaysia, Malaysia, and Universiti Sains Malaysia,

Malaysia.

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